Methods of administering adenoviral vectors

ABSTRACT

The present invention provides methods for administering an adenoviral gene transfer vector comprising an exogenous gene to an animal. One method involves utilizing systemic neutralizing antibodies to neutralize the adenoviral gene transfer vector outside a targeted muscle. Another method involves the repeat administration of an adenoviral gene transfer vector to a skeletal muscle.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to methods useful in theadministration of gene products to animals using adenoviral vectors.

BACKGROUND OF THE INVENTION

[0002] Modified viruses have proven convenient vector systems forinvestigative and therapeutic gene transfer applications, and adenoviralvector systems present several advantages for such uses. Adenovirusesare generally associated with benign pathologies in humans, and the 36kb of the adenoviral genome has been extensively studied. Adenoviralvectors can be produced in high titers (e.g., about 10¹³ pfu), and suchvectors can transfer genetic material to nonreplicating, as well asreplicating, cells (in contrast with, for example, retroviral vectorswhich only transfer genetic material to replicating cells). Theadenoviral genome can be manipulated to carry a large amount ofexogenous DNA (up to about 8 kb), and the adenoviral capsid canpotentiate the transfer of even longer sequences (Curiel et al., Hum.Gene Ther., 3, 147-154 (1992)). Additionally, adenoviruses generally donot integrate into the host cell chromosome, but rather are maintainedas a linear episome, thus minimizing the likelihood that a recombinantadenovirus will interfere with normal cell function. Aside from being asuperior vehicle for transferring genetic material to a wide variety ofcell types, adenoviral vectors represent a safe choice for genetransfer, a particular concern for therapeutic applications.

[0003] A variety of recombinant adenoviral vectors have been described.Most of the vectors in use today derive from the adenovirus serotype 5(Ad5), a member of subgroup C. An exogenous gene of interest typicallyis inserted into the early region 1 (E1) of the adenovirus. Disruptionof the E1 region decreases the amount of viral proteins produced by boththe early regions (DNA binding protein) and late regions (penton, hexon,and fiber proteins), preventing viral propagation. These replicationdeficient adenoviral vectors require growth in either a complementarycell line or in the presence of an intact helper virus, which provides,in trans, the essential E1 functions (Berker et al., J. Virol., 61,1213-1220 (1987); Davidson et al., J. Virol., 61, 1226-1239 (1987);Mansour et al., Mol. Cell Biol., 6, 2684-2694 (1986)). More recently,adenoviral vectors deficient in both E1 and the early region 4 (E4) havebeen used to substantially abolish expression of viral proteins. Inorder to insert the larger genes (up to 8 kb) into the adenoviralgenome, adenoviral vectors additionally deficient in the nonessentialearly region 3 (E3) are used. Multiply deficient adenoviral vectors aredescribed in published PCT patent application WO 95/34671.

[0004] One limitation of adenoviral vector systems is the ability of theadenoviral vector to transduce a wide variety of proliferating andquiescent cells (Michou et al., Gene Ther., 4, 473-482 (1997)). Thisability, while a benefit in transducing the target area, is a limitationwhen the adenoviral vector “leaks” out of the targeted area andtransduces other cells it contacts. Tranduction of the surrounding cellsis a severe problem when the gene product encoded by the adenoviralvector is harmful, toxic, or otherwise undesirable with respect to thesenon-targeted areas.

[0005] Another limitation of the adenoviral vector system is thecellular and humoral immune response generated within the host animal.Initial administration elicits a reaction from both CD8⁺ and CD4⁺ T celllymphocytes which eliminate virus infected cells within 28 days afterinfection, limiting the duration of the transgene expression. Inaddition, neutralizing antibodies produced by B lymphocytes incooperation with CD4⁺ cells inhibit the effectiveness of a repeatadministration of the adenoviral vector. Proliferation and specificityof the antibodies is achieved through interactions between theadenoviral vector, B-cell surface immunoglobulins and activated CD4⁺surface proteins (particularly CD40Li, which binds CD40 on the surfaceof the B cell) (Yang et al., J. Virol., 69, 2004 (1995)).

[0006] Attempts to circumvent the humoral immune response to allowrepeat administration of the adenoviral vector have met with limitedsuccess. These attempts have been focused in two areas,immunosuppression and alteration of the adenoviral vector. Severalgroups have experimented with various immunosuppressant drugs orantibodies specific for CD4⁺, CD40 ligand, or CTLA4Ig to reduce theadenovirus-specific humoral immune response (Lee et al., Hum. GeneTher., 7, 2273 (1996) (CD4⁺); Yang et al., J. Virol., 70, 6370 (1996)(CD40 ligand); Kay et al., Nature Gen., 11, 191 (1995) (CTLA4Ig)).Although some of these results have been encouraging, there is asubstantial risk associated with systemic immune suppression in aclinical setting.

[0007] In another study, subretinal administration of an adenoviralvector containing the bacterial β-galactosidase gene resulted in minimalcirculating antibodies specific to the adenoviral vector. This was mostlikely a reflection of the immune privileged status of the retina.Although there was minimal retinal toxicity to the adenovirus, severalof the animals injected developed localized granulomatous infiltrate atthe injection site (Bennett et al., Hum. Gene Ther., 7, 1763-1769(1996)). Subretinal administration is not an option for manyapplications where adenoviral vectors are employed.

[0008] Alteration of the adenoviral vector is time consuming and has notbeen entirely successful in sufficiently attenuating the immuneresponse. Limited readministration of the adenoviral vector has beenaccomplished when adenoviral vectors of different serotypes within thesame subgroup are used; however, persistence of expression of thetransgene was not comparable to the initial administration (Mack et al.,Hum. Gene Ther., 8, 99-109 (1997)).

[0009] Accordingly, there is a need for improved methods ofadministering adenoviral vectors to animals, particularly, to preventleakage of the adenoviral vector from the target area and to circumventthe humoral immune response elicited by adenoviral vectors. The presentinvention provides such methods. This and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides a method of targeting a geneproduct in a particular muscle of an animal. The method utilizessystemic neutralizing antibodies to neutralize an adenoviral genetransfer vector containing an exogenous gene outside the particularmuscle. The adenoviral gene transfer vector is administered such thatthe exogenous gene is expressed and the gene product is produced only inthe particular muscle of administration.

[0011] The present invention further provides a method of producing agene product in a skeletal muscle of an animal. The method comprises afirst intramuscular administration of an adenoviral vector to theskeletal muscle of an animal, and a second administration of anadenoviral gene transfer vector containing an exogenous gene encoding agene product. Administration is such that the exogenous gene isexpressed and the gene product is produced in the skeletal muscle of theanimal.

[0012] The invention may best be understood with reference to theaccompanying drawings and in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a schematic diagram depicting the original adenovirusused to derive the adenoviral vectors AdCMVNull and AdCMV.Z, the regionsof addition and deletion of the original adenovirus, and the expressioncassettes of the adenoviral vectors AdCMVNull and AdCMV.Z.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides methods useful in theadministration of gene products to animals using adenoviral genetransfer vectors. The ability to target an adenoviral vector and torepeatedly administer a therapeutic adenoviral vector in a clinicalsetting is useful in improving treatment efficacy and in enabling thetreatment of diseases. This invention provides a method to limit theinfection of non-target tissue following administration of an adenoviralvector to a particular muscle of an animal. The vector targetingpotential is useful for cardiac, particularly, endocardial,administration, as the risk of misinjection of the adenoviral vector ishigh. As adenoviral vectors cannot be readministered systemically, thepresent invention also provides a method for repeat administration of anadenoviral gene transfer vector comprising an exogenous gene to theskeletal muscle of an animal.

[0015] The term “exogenous gene”, as it is used herein, refers to anygene in an adenoviral gene transfer vector which is not native to theadenovirus which comprises the adenoviral vector. The gene includes anucleic acid sequence encoding a gene product operably linked to apromoter. Any portion of the gene can be non-native to the adenoviruswhich comprises the adenoviral vector. For example, the gene cancomprise a non-native nucleic acid sequence encoding a gene productwhich is operably linked to a native promoter. It should be appreciatedthat the exogenous gene can be any gene encoding an RNA or protein ofinterest to the skilled artisan. Therapeutic genes, genes encoding aprotein that is to be studied in vitro and/or in vivo, genes encodinganti-sense RNA's, and modified viral genes are illustrative of possibleexogenous genes.

[0016] The term “adenoviral gene transfer vector”, as it is used herein,refers to any replication incompetent adenoviral vector with anexogenous gene encoding a gene product inserted into its genome. Thevector must be capable of replicating and being packaged when anydeficient essential genes are provided in trans. An adenoviral vectordesirably contains at least a portion of each terminal repeat requiredto support the replication of the viral DNA, preferably at least about90% of the full ITR sequence, and the DNA required to encapsidate thegenome into a viral capsid. Many suitable adenoviral vectors have beendescribed in the art.

[0017] In one embodiment, the present invention provides a method oftargeting a gene product to a muscle of an animal using an adenoviralgene transfer vector containing an exogenous gene encoding a geneproduct. Systemic neutralizing antibodies to a particular adenoviralgene transfer vector are first induced in the animal. The adenoviralvector is then administered to a particular muscle of an animal suchthat the exogenous gene encoded by the adenoviral vector is expressedand the gene product produced in the particular muscle of the animal. Inaddition, the adenoviral vector is neutralized outside the muscle ofadministration.

[0018] The present invention can be practiced with any suitable animal,preferably a mammal, more preferably, a human. Additionally, theadenoviral vector can be administered to any suitable muscle of theanimal; however, it is preferably administered to the heart.

[0019] Any suitable method can be used to induce systemic neutralizingantibodies to the adenoviral vector. Desirably, an antigen isadministered to the animal. This antigen can be the adenoviral genetransfer vector, but preferably, it is an identical adenoviral vector,except without an exogenous gene (an AdCMVNull vector, an example ofwhich can be found in FIG. 1). The antigen can also be administered byany suitable method. Depending on the antigen, administration can be toany suitable area of the animal. In order to induce the systemicneutralizing antibodies, the antigen can be administered any number ofsuitable times, e.g., once, twice, or more.

[0020] Using the AdCMVNull vector administration, the antigen can beadministered systemically (rather than to the target muscle) to preventany damage to the particular muscle. Systemic administration can beaccomplished through intravenous injection, either bolus or continuous,or any other suitable method. An added benefit of systemicadministration is that it requires a much smaller amount of antigen toproduce the same levels of circulating antibodies as administration toany muscle of the animal.

[0021] Administration of the antigen produces circulating neutralizingantibodies. While not wishing to be bound by any particular theory, itis believed that when the adenoviral gene transfer vector isadministered to the particular muscle of the animal, some of theadenoviral particles escape the muscle. These adenoviral particles arethen neutralized by the antibodies circulating throughout the animalsuch that significantly less (and preferably substantially no) geneproduct is produced outside the particular muscle. The amount ofexogenous gene product produced outside the area of administration ispreferably at least 10% less (more preferably at least 50% less, andmost preferably at least 80% less) than production of the gene productoutside the particular muscle of administration in a naive animal, whichdoes not have circulating neutralizing antibodies to the adenoviral genetransfer vector.

[0022] Neutralization of adenoviral particles outside of the particularmuscle prevents production of the exogenous gene carried in theadenoviral gene transfer vector. This is extremely useful in situationswhere the exogenous gene is harmful, or toxic, to the animal whenpresent in areas other than the particular muscle of administration. Anexample of this is vascular endothelial growth factor (VEGF protein),which mediates vascular growth. While vascular growth is desirable inthe heart to repair damaged cardiac muscle, growth outside the heart canlead to severe problems, including blindness, and increasedaggressiveness of tumor cells.

[0023] In another embodiment, the present invention provides a method ofproducing a gene product in a skeletal muscle. An adenoviral vector isfirst administered to the skeletal muscle of an animal. An adenoviralvector containing an exogenous gene encoding a gene product is thenadministered to the same skeletal muscle such that the exogenous gene isexpressed and the gene product is produced in the skeletal muscle. Anysuitable animal can be used; however, preferably, the animal is amammal, more preferably, a human.

[0024] After the second or subsequent administration of the adenoviralgene transfer vector, production of the gene product in the muscle ofthe animal is desirably at least 1% of (such as at least 10% of,preferably at least 50% of, more preferably at least 80% of, and mostpreferably, substantially the same as) production of the gene productafter a first or preceding administration with the same adenoviral genetransfer vector containing the exogenous gene encoding the gene product.While not wishing to be bound by any particular theory, it is believedthat the level of gene product produced in the skeletal muscle of ananimal after the second or subsequent administration to the muscle canbe substantially similar to that of the first or precedingadministration because neutralizing antibodies, which are produced bythe first or preceding administration, cannot readily penetrate themuscle and destroy the adenoviral gene transfer vector. This holds trueeven when the neutralizing antibody response is boosted with two or moreinitial administrations before the final or subsequent intramuscularadministration of the adenoviral gene transfer vector containing theexogenous gene encoding the gene product.

[0025] To facilitate the administration of adenoviral vectors, they canbe formulated into suitable pharmaceutical compositions. Generally, suchcompositions include the active ingredient (i.e., the adenoviral vector)and a pharmacologically acceptable carrier. Such compositions can besuitable for delivery of the active ingredient to a patient for medicalapplication, and can be manufactured in a manner that is itself known,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

[0026] Pharmaceutical compositions for use in accordance with thepresent invention can be formulated in a conventional manner using oneor more pharmacologically or physiologically acceptable carrierscomprising excipients, as well as optional auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Thus, for injection, the active ingredient can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. For oral administration,the active ingredient can be combined with carriers suitable forinclusion into tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like. For administration by inhalation,the active ingredient is conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant. The active ingredient can beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Such compositions can take such formsas suspensions, solutions or emulsions in oily or aqueous vehicles, andcan contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Other pharmacological excipients are known in theart.

[0027] The present inventive methods are useful in the context of thetreatment of animals, e.g., medical treatment. In addition, the presentinventive methods are useful in the production of gene products, e.g.,in vivo protein production (which can entail subsequent proteinrecovery) as well as in research, e.g., investigation of geneexpression, adenoviral targeting, and the like.

EXAMPLE

[0028] The present invention is further described in the followingexample. This example serves only to illustrate the invention and is notintended to limit the scope of the invention in any way.

[0029] This example illustrates use of the present inventive method oftargeting production of a gene product to a particular muscle in ananimal, as well as the present inventive method of repeat administrationto produce a gene product in a skeletal muscle of an animal. Inparticular, systemic neutralizing antibodies to an adenoviral vectorwere induced in an animal, and then the adenoviral vector comprising anexogenous gene encoding a gene product was administered to a particularmuscle of the animal such that the exogenous gene was expressed and thegene product was produced in the particular muscle of the animal. Inaddition, the adenoviral vector was neutralized outside of theparticular muscle of the animal such that there was limited expressionof the exogenous gene resulting in production of the gene productoutside of the particular muscle of the animal.

[0030] For the purposes of this experimental work, C57B16 mice were usedas the test animals because their immune system is able to recognizeadenoviral vectors as foreign antigens and mount a sufficient immuneresponse to destroy the adenoviral vectors, thereby preventingexpression of an exogenous gene forming a part of the adenoviral vector.The mice were separated into three groups. Systemic neutralizingantibodies were induced in the mice of group 1 with an adenoviral vectorwhich did not contain an exogenous gene encoding a gene product(AdCMVNull). A similar adenoviral vector (AdCMV.Z), with a geneexpression cassette encoding a reporter gene product (i.e., a geneproduct that could be readily detected), was administered to the mice ofgroups 1 and 2 to determine whether production of the reporter geneproduct β-galactosidase (β-gal) was limited to the right gastrocnemiusmuscle or could be detected in other areas of the mice, particularly theliver inasmuch as adenoviral vectors are known to localize in the liverafter entering the bloodstream of an animal (Jaffee et al., Nat. Genet.,1, 372-78 (1992)). The mice of group 2 were treated as a naive group.Only the adenoviral vector AdCMV.Z, with a gene expression cassetteencoding the reporter gene product β-gal, was administeredintrajugularly to the mice of group 2, i.e., no adenoviral vector wasadministered to induce systemic neutralizing antibodies in the micebefore the administration of the adenoviral vector AdCMV.Z. The mice ofgroup 2 otherwise were treated in the same manner as the mice ofgroup 1. Finally, a control group, group 3, which did not receive anyadministration of adenoviral vectors, was included.

[0031] The AdCMVNull vector was a replication-deficient adenoviralvector with deletions in the E1 and E3 regions. An expression cassettewas inserted in the E1-deleted region of the adenoviral vector thatincluded an SV40 polyA sequence and a cytomegaloviral promoter (CMV).The AdCMVNull vector is depicted in FIG. 1.

[0032] The AdCMV.Z vector was also a replication-deficient adenoviralvector similar to the AdCMVNull vector, except that the expressioncassette included a nucleic acid sequence encoding the reporter geneproduct β-gal operably linked to the CMV promoter, from left to right,relative to the viral vector. The AdCMV.Z vector is also depicted inFIG. 1.

[0033] The protocol for administration of the AdCMVNull and AdCMV.Zvectors to the mice of the two groups was as follows: the mice of group1 were immunized with an intramuscular injection of 1×10¹⁰ pu ofAdCMVNull on day 1 of the experiment, and received a subsequentintramuscular injection of 1×10¹⁰ pu of AdCMV.Z on day 14. The mice ofgroup 2 (the naive mice) received an injection of 1×10¹⁰ pu of AdCMV.Zon day 14. The mice of group 3 did not receive any injections.

[0034] On day 15, the mice in all three groups were sacrificed. Theβ-gal activity in the mice was determined in the liver and rightgastrocnemius muscle. Neutralizing antibody titers also were determinedin the mice. The results of these analyzes are set forth below inTable 1. TABLE 1 β-galactosidase Neutralizing Activity (RLU/mg protein)Antibodies Right Gastrocnemius (reciprocal Muscle Liver dilution) Group1 1.4447 × 10⁶ 8.0697 × 10³ 32 (AdCMVNull) Group 2 4.0748 × 10⁶ 5.2022 ×10⁶ 1.0 (Naive) Group 3 1.0683 × 10⁴  7.898 × 10³ n/a (Control)

[0035] As is apparent from the experimental results set forth above, themice in the first two groups had essentially the same levels of β-galactivity in the right gastrocnemius muscle, about 10⁶ RLU/mg. The miceof group 3 (the control group) had a β-gal activity level of about 10⁴RLU/mg. The results demonstrate that there was gene expression in thetargeted muscle, even in the mice of group 1, which were the subject ofthe repeat administration. Moreover, the mice of group 1, in whichsystemic neutralizing antibodies were induced, had significantly lessβ-gal activity in the liver, about 10⁴ (or a hundred-fold less thanmeasured in the target muscle and approximately the same as thecontrol), thereby demonstrating that there was localization of thetargeted gene product to the targeted muscle in accordance with thepresent invention. In distinct contrast, the mice of group 2, in whichneutralizing antibodies were not induced, had essentially the same levelof β-gal activity in the liver, about 10⁶ RLU/mg, as in the targetedmuscle, thereby indicating that in the absence of the present inventivemethod, when there is undesirable leaking of the adenoviral vectoroutside the targeted muscle, there is wide-spread production of the geneproduct of interest.

[0036] All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

[0037] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations of the preferred embodiments may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims below.

What is claimed is:
 1. A method of targeting a gene product to aparticular muscle of an animal, said method comprising (a) inducing inan animal systemic neutralizing antibodies to an adenoviral genetransfer vector, (b) administering said adenoviral gene transfer vectorcomprising an exogenous gene encoding a gene product to a particularmuscle of said animal such that said exogenous gene is expressed andsaid gene product is produced in said particular muscle of said animal,and (c) neutralizing said adenoviral gene transfer vector outside ofsaid particular muscle of said animal.
 2. The method of claim 1 ,wherein said animal is a mammal.
 3. The method of claim 2 , wherein saidmammal is a human.
 4. The method of claim 1 , wherein said systemicneutralizing antibodies to said adenoviral gene transfer vector areproduced by administration of an antigen.
 5. The method of claim 4 ,wherein said antigen is the same as said adenoviral gene transfervector, except that it does not contain an exogenous gene encoding agene product.
 6. The method of claim 4 , wherein said antigen is thesame as said adenoviral gene transfer vector.
 7. The method of claim 4 ,wherein said antigen is administered to said animal systemically.
 8. Themethod of claim 1 , wherein said neutralizing of said adenoviral genetransfer vector outside said particular muscle of said animal is aresult of the presence of said neutralizing antibodies.
 9. The method ofclaim 1 , wherein said neutralizing of said adenoviral gene transfervector outside said particular muscle of said animal is such that saidproduction of said gene product is at least 90% less than the productionof said gene product outside said particular muscle of a naive animal ofthe same species as said animal after administration of said adenoviralgene transfer vector.
 10. The method of claim 9 , wherein saidneutralizing of said adenoviral gene transfer vector outside saidparticular muscle of said animal is such that said production of saidgene product is at least 99% less than the production of said geneproduct outside said particular muscle of a naive animal of the samespecies as said animal after administration of said adenoviral genetransfer vector.
 11. The method of claim 10 , wherein said neutralizingof said adenoviral gene transfer vector outside said particular muscleof said animal is such that said production of said gene product is atleast 99.9% less than the production of said gene product outside saidparticular muscle of a naive animal of the same species as said animalafter administration of said adenoviral gene transfer vector.
 12. Amethod of producing a gene product in a skeletal muscle of an animal,said method comprising (a) administering an adenoviral vector to saidskeletal muscle of said animal, and (b) at least seven days after saidadministration, administering an adenoviral gene transfer vectorcomprising an exogenous gene encoding a gene product to said skeletalmuscle of said animal such that said exogenous gene is expressed andsaid gene product is produced in said skeletal muscle of said animal.13. The method of claim 12 , wherein said animal is a mammal.
 14. Themethod of claim 13 , wherein said mammal is a human.
 15. The method ofclaim 12 , wherein said adenoviral vector in step (a) is said adenoviralgene transfer vector comprising an exogenous gene encoding a geneproduct in step (b).
 16. The method of claim 15 , wherein production ofsaid gene product in said skeletal muscle of said animal as a result ofsaid second intramuscular administration of said adenoviral genetransfer vector is at least 10% of the production of said gene productas a result of said first intramuscular administration of saidadenoviral gene transfer vector comprising an exogenous gene encoding agene product.
 17. The method of claim 16 , wherein production of saidgene product in said skeletal muscle of said animal as a result of saidsecond intramuscular administration of said adenoviral gene transfervector is at least 50% of the production of said gene product as aresult of said first intramuscular administration of said adenoviralgene transfer vector comprising an exogenous gene encoding a geneproduct.
 18. The method of claim 17 , wherein production of said geneproduct in said skeletal muscle of said animal as a result of saidsecond intramuscular administration of said adenoviral gene transfervector is at least 80% of the production of said gene product as aresult of said first intramuscular administration of said adenoviralgene transfer vector comprising an exogenous gene encoding a geneproduct.
 19. The method of claim 18 , wherein production of said geneproduct in said skeletal muscle of said animal as a result of saidsecond intramuscular administration of said adenoviral gene transfervector is at least substantially the same as the production of said geneproduct as a result of said first intramuscular administration of saidadenoviral gene transfer vector comprising an exogenous gene encoding agene product.